I am trying to improve our assays to immunophenotyping blood cells using 96-well plates. I've tried several dilutions of FACS Lysing solution in 50 uL of whole blood however it did not work. So if someone performs red blood lysing in 96-well and could share the protocol I will be thankful.
I am using RBC lysis buffer (10x)from biolegend. i usually lysis 5ml for even 50 million cells. 5-10 min. it works very well for spleen cells. I have no experience for whole blood. If you stain first and lysis later, maybe shaking is better for lysis? If you lysis first and stain later, maybe you ccould do bulk lysis before distrubuting to single wells.
Hi Paola!
I would suggest lo lyse RBCs before dividing WBCs in wells. I usually do it this way and works well. I use Miltenyi RBC Lysis Buffer following instructions: dilute the buffer 1:10 in distilled water and add it 1:10 to blood (which means, if you have 5ml of blood, you should add 45ml of buffer. However, if you pellet your cells, usually 5-10ml are fine). I recommend to be careful that distilled water you use is actually pure. Few weeks ago my buffer suddenly stopped working and I discovered it was due to water. So, if yours still does not work, try and change the distilled water you use
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